Error detection in chemical array fabrication

ABSTRACT

A method, apparatus, and computer program product, for forming an addressable array of chemical moieties on a substrate. The method may include, for each of multiple locations on the substrate, depositing a reagent drop set during a cycle so as to attach a corresponding moiety for that location. This may be repeated as required, until the addressable array is formed. Test location are also formed by depositing drops from dispensers in a manner which can facilitate detection of dispenser errors.

FIELD OF THE INVENTION

This invention relates to arrays, particularly polynucleotide arrayssuch as DNA arrays, which are useful in diagnostic, screening, geneexpression analysis, and other applications.

BACKGROUND OF THE INVENTION

In the following discussion and throughout the present application, nocited reference is admitted to be prior art to the present application.

Polynucleotide arrays (such as DNA or RNA arrays), are known and areused, for example, as diagnostic or screening tools. Such arrays includeregions of usually different sequence polynucleotides arranged in apredetermined configuration on a substrate. These regions (sometimesreferenced as “features”) are positioned at respective locations(“addresses”) on the substrate. The arrays, when exposed to a sample,will exhibit an observed binding pattern. This binding pattern can bedetected upon interrogating the array. For example all polynucleotidetargets (for example, DNA) in the sample can be labeled with a suitablelabel (such as a fluorescent compound), and the fluorescence pattern onthe array accurately observed following exposure to the sample. Assumingthat the different sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more polynucleotide components of the sample.

Biopolymer arrays can be fabricated by depositing previously obtainedbiopolymers (such as from synthesis or natural sources) onto asubstrate, or by in situ synthesis methods. Methods of depositingobtained biopolymers include loading then touching a pin or capillary toa surface, such as described in U.S. Pat. No. 5,807,522 or deposition byfiring from a pulse jet such as an inkjet head, such as described inU.S. Pat. No. 6,180,351, PCT publications WO 95/25116 and WO 98/41531,and elsewhere. Such a deposition method can be regarded as forming eachfeature by one cycle of attachment (that is, there is only one cycle ateach feature during which the previously obtained biopolymer is attachedto the substrate). For in situ fabrication methods, multiple differentreagent droplets are deposited by pulse jet or other means at a giventarget location in order to form the final feature (hence a probe of thefeature is synthesized on the array substrate). The in situ fabricationmethods include those described in U.S. Pat. No. 5,449,754 forsynthesizing peptide arrays, and in U.S. Pat. No. 6,180,351 and WO98/41531 and the references cited therein for polynucleotides, and mayalso use pulse jets for depositing reagents. The in situ method forfabricating a polynucleotide array typically follows, at each of themultiple different addresses at which features are to be formed, thesame conventional iterative sequence used in forming polynucleotidesfrom nucleoside reagents on a support by means of known chemistry. Thisiterative sequence can be considered as multiple ones of the followingattachment cycle at each feature to be formed: (a) coupling an activatedselected nucleoside (a monomeric unit) through a phosphite linkage to afunctionalized support in the first iteration, or a nucleoside bound tothe substrate (i.e. the nucleoside-modified substrate) in subsequentiterations; (b) optionally, blocking unreacted hydroxyl groups on thesubstrate bound nucleoside (sometimes referenced as “capping”); (c)oxidizing the phosphite linkage of step (a) to form a phosphate linkage;and (d) removing the protecting group (“deprotection”) from the nowsubstrate bound nucleoside coupled in step (a), to generate a reactivesite for the next cycle of these steps. The coupling can be performed bydepositing drops of an activator and phosphoramidite at the specificdesired feature locations for the array. Capping, oxidation anddeprotection can be accomplished by treating the entire substrate(“flooding”) with a layer of the appropriate reagent. The functionalizedsupport (in the first cycle) or deprotected coupled nucleoside (insubsequent cycles) provides a substrate bound moiety with a linkinggroup for forming the phosphite linkage with a next nucleoside to becoupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, inanother flooding procedure in a known manner. Conventionally, a singlepulse jet or other dispenser is assigned to deposit a single monomericunit.

The foregoing chemistry of the synthesis of polynucleotides is describedin detail, for example, in Caruthers, Science 230: 281-285, 1985;Itakura et al., Ann. Rev. Biochem. 53: 323- 356; Hunkapillar et al.,Nature 310: 105-110, 1984; and in “Synthesis of OligonucleotideDerivatives in Design and Targeted Reaction of OligonucleotideDerivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S.Pat. No. 5,869,643, EP 0294196, and elsewhere The phosphoramidite andphosphite triester approaches are most broadly used, but otherapproaches include the phosphodiester approach, the phosphotriesterapproach and the H-phosphonate approach. The substrates are typicallyfunctionalized to bond to the first deposited monomer. Suitabletechniques for functionalizing substrates with such linking moieties aredescribed, for example, in Southern, E. M., Maskos, U. and Elder, J. K.,Genomics, 13, 1007-1017, 1992.

In the case of array fabrication, different monomers and activator maybe deposited at different addresses on the substrate during any onecycle so that the different features of the completed array will havedifferent desired biopolymer sequences. One or more intermediate furthersteps may be required in each cycle, such as the conventional oxidation,capping and washing steps in the case of in situ fabrication ofpolynucleotide arrays (again, these steps may be performed in floodingprocedure).

In array fabrication, the quantities of polynucleotide available areusually very small and expensive. Additionally, sample quantitiesavailable for testing are usually also very small and it is thereforedesirable to simultaneously test the same sample against a large numberof different probes on an array. These conditions require use of arrayswith large numbers of very small, closely spaced features. It isimportant in such arrays that features actually be present, that theyare put down as accurately as possible in the desired target pattern,are of the correct size, and that the DNA is uniformly coated within thefeature. If any of these conditions are not met within a reasonabletolerance, the results obtained from a given array may be unreliable andmisleading. This of course can have serious consequences to diagnostic,screening, gene expression analysis or other purposes for which thearray is being used.

However, in any system used to fabricate arrays with the required smallfeatures, there is inevitably some degree of error, either fixed (andhence repeated) and/or random. In the case of both the deposition ofpreviously obtained biopolymers, but particularly in the in situfabrication method, drop deposition errors may occur from cycle to cycleand may be different from different cycles or may be the same. Forexample, one or more drop dispensers may exhibit an error (that is, amalfunction) during particular cycles or may have a same malfunctionover multiple cycles. Such malfunctions include errors in firing (thatis, “misfiring” which includes not dispensing a drop at all ordispensing an incorrect drop volume) as well as trajectory errors (thatis, the dispenser fires a drop on an unexpected error due, for example,to an error in nozzle construction).

It would be desirable then to provide a means by which drop dispensererrors during any array fabrication method, can be identified.

SUMMARY OF THE INVENTION

The present invention realizes that drop dispenser errors may beevaluated by detecting (such as by imaging) one or more drops depositedby the dispenser onto the substrate during array fabrication. However,the present invention further realizes that multiple dispensers maydeposit drops at a same feature over one or more same cycles. In thissituation, while a first drop deposited by a first dispenser on asubstrate location may be detected, it is difficult to detect a secondand subsequent drops deposited at the same location by differentdispensers. This is so in the case since where different dispensersdispense drops to the same location during a same cycle, the second andsubsequent drops mix with the first drop previously deposited on thesubstrate and it may be difficult to then evaluate whether the second orsubsequent drops were actually deposited or of correct volume andlocation (that is, it may be difficult to evaluate whether the secondand subsequent drop dispensers are in error). Other factors may alsomake such an evaluation difficult. Even in the case where only one dropis dispensed by a dispenser during each of multiple cycles, withdifferent drops being dispensed by different dispensers at the samelocation over multiple cycles, it may become difficult to detect laterdrops. For example, changes in surface properties of the substrate mayoccur as a result of one or more reagents (such as an activatedphosphoramidite) being applied to the same location over the manycycles. In this case, the feature location on the substrate becomes morehydrophilic than the hydrophobic starting substrate surface. In such asituation even though a dispenser may dispense a drop slightly off froma target location (for example 20 μm off the center of a feature) thedroplet may still migrate to the center because it prefers thehydrophilic region, and the resulting array is fine. Thus, it may beuseful to evaluate the results from the detecting based on what cyclethey are from in order to estimate their impact. A same deposition error(for example, the 20 μm error mentioned) at earlier cycles in the startof a feature synthesis will likely be of more concern than at latercycles.

The present invention then, provides in a method of forming anaddressable array of chemical moieties on a substrate. The methodincludes for each of multiple locations (sometimes referenced as“feature locations”) on the substrate, depositing a reagent drop setduring a cycle so as to attach a corresponding moiety for that location.The foregoing is repeated as required, until the addressable array isformed. In any event, for each of multiple locations, a multi-dispenserdrop group is deposited over one or more cycles for a correspondingfeature location which group includes drops which are deposited fromdifferent dispensers. By “over” in this context is meant in one cycle,or as a result of multiple cycles (for example, two dispenser onedispenses one drop and dispenser two dispenses another drop at the samelocation) The method additionally includes depositing and detectingdrops at respective separate locations (sometimes referenced as “testlocations”) on the substrate from different dispensers which deposit amulti-dispenser drop group.

The present invention also provides a method of forming multipleaddressable arrays of chemical moieties on a substrate, in which foreach array, for each of multiple feature locations on the substrate areagent drop set is deposited during a cycle so as to attach acorresponding moiety for that location. This step is repeated ifrequired until the addressable array is formed. Multiple dispensers areused to dispense drops to form the array. The method additionallyincludes depositing and detecting drops from the different dispensers atrespective separate locations on the substrate, wherein the drops aredeposited at a separate test pattern area between arrays. In this case,the number of test locations of the test pattern area during any onecycle may be less than one quarter (or even less than one-tenth orone-twentieth) the number of locations in the smallest of the arrayswhich the test pattern area is between. Also, the number of locations ofthe test pattern area during any one cycle may be such as to not begreater than ten times (or not greater than five or two times, and mayeven be the same as) the number of the dispensers used to form an arrayduring any one cycle. The test pattern area may be the same area throughwhich the substrate is later cut or otherwise separated to providemultiple portions of the substrate each with one or more fabricatedarrays thereon.

The method may be used for forming an addressable array of polymers onthe substrate. In this case, the reagent drop set deposited during acycle attaches a monomeric unit of the corresponding polymer for thatfeature location. However, a method of the present invention could alsobe used

A method of the present invention may (by “may” throughout thisapplication is meant “optionally”) include the possibility of notindependently detecting drops of a drop group at a corresponding featurelocation at which such group is deposited, for one or more or all suchfeature locations receiving a drop group. Alternatively, oradditionally, a method of the present invention may include thereferenced separate test locations in a test pattern area which isseparate from (that is, distinguishable from) the array.

A method of the present invention may use a multi-dispenser drop groupwhich has a drop including an attachment moiety which becomes attachedat a feature location at which the group is deposited but which does notbecome attached at test location. For example, the attachment moiety maybe one which will become attached at the feature location uponactivation by an activator. Another drop of the group may include theactivator moiety. In the foregoing example, the attachment moiety andactivator are deposited at separate test locations. Results from thedetecting (such as an image) may be evaluated for a dispenser error.When an error is detected, the array may be discarded or an attempt madeto correct such error (such as by depositing further drops to correctthe error), or the results used for other purposes. Also, drops of amulti-dispenser drop group may, but need not, contact one another. Forexample, in the attachment moiety and activator combination alreadymentioned, drops of each will typically be deposited in a same cycle andso will contact one another. However, particularly where themulti-dispenser drop group is over multiple cycles, a drop of the groupdeposited in one cycle may already have been removed from thecorresponding feature location before a drop of the group in anothercycle contacts that same feature location.

The present invention further provides an apparatus which an execute amethod of the present invention. Such an apparatus may include adeposition system having multiple dispensers each of which can dispensea reagent drop, as well as a transport system to move at least one ofthe deposition system or the substrate. A drop detector may further bepresent, as well as a process which controls the deposition system andthe transport system such that a method of the present application maybe performed. The present invention also provides a computer programproduct which can execute any one or more methods of the presentinvention. Optionally, the present invention may further provide forexposing the array to a sample, and reading the array following theexposure and optionally processing results of the interrogation. Theresult of an array reading (whether processed or not) may be forwardedfor receipt at a remote location.

The various aspects of the present invention can provide any one or moreof the following and/or other useful benefits. For example, a dispensererror which could result in a feature error during an in situ or anyarray fabrication method, can be detected. This allows an opportunity toidentify and discard possibly defect arrays or an opportunity to attemptto correct the defect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a substrate carrying multiple arrays, such as may befabricated by methods of the present invention;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing multiple idealspots or features;

FIG. 3 is an enlarged illustration of a portion of the substrate in FIG.2;

FIG. 4 is a top view of orifices of drop dispensers;

FIG. 5 schematically illustrates a pattern of deposited drops over twocycles for fabrication of the first two nucleotides of the array portionillustrated in FIG. 3;

FIG. 6 illustrates a test pattern as formed during a method of thepresent invention;

FIG. 7 illustrates multiple arrays and multiple test patterns on asubstrate, as may be fabricated and used during methods of the presentinvention;

FIG. 8 is a top view of orifices on a particular deposition head andpart of a corresponding test pattern which may be deposited according toa method of the present invention; and

FIG. 9 schematically illustrates an apparatus of the present invention.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate identical elements that are commonto the figures.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In the present application, unless a contrary intention appears, thefollowing terms refer to the indicated characteristics. A “biopolymer”is a polymer of one or more types of repeating units. Biopolymers aretypically found in biological systems and particularly includepolysaccharides (such as carbohydrates), and peptides (which term isused to include polypeptides and proteins) and polynucleotides as wellas their analogs such as those compounds composed of or containing aminoacid analogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. This includes polynucleotides in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids (or synthetic or naturallyoccurring analogs) in which one or more of the conventional bases hasbeen replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions.Polynucleotides include single or multiple stranded configurations,where one or more of the strands may or may not be completely alignedwith another. A “nucleotide” refers to a sub-unit of a nucleic acid andhas a phosphate group, a 5 carbon sugar and a nitrogen containing base,as well as functional analogs (whether synthetic or naturally occurring)of such sub-units which in the polymer form (as a polynucleotide) canhybridize with naturally occurring polynucleotides in a sequencespecific manner analogous to that of two naturally occurringpolynucleotides. For example, a “biopolymer” includes DNA (includingcDNA), RNA, oligonucleotides, and PNA and other polynucleotides asdescribed in U.S. Pat. No. 5,948,902 and references cited therein (allof which are incorporated herein by reference), regardless of thesource. An “oligonucleotide” generally refers to a nucleotide multimerof about 10 to 100 nucleotides in length, while a “polynucleotide”includes a nucleotide multimer having any number of nucleotides. A“biomonomer” references a single unit, which can be linked with the sameor other biomonomers to form a biopolymer (for example, a single aminoacid or nucleotide with two linking groups one or both of which may haveremovable protecting groups). A biomonomer fluid or biopolymer fluidreference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

A “drop” is a small amount of liquid traveling in a space, and whileoften approximately spherical if no external forces are acting upon it,may have other shapes depending upon those other forces. In the presentcase, a drop which has contacted a substrate is often referred to as adeposited drop, although sometimes it will be simply referenced as adrop when it is understood that it was previously deposited. Detecting adrop “at” a location, includes the drop being detected while it istraveling between a dispenser and that location, or after it hascontacted that location (and hence may no longer retain its originalshape) such as capturing an image of a drop on the substrate after ithas assumed an approximately circular shape of a deposited drop. A“pulse jet” is a device which can dispense drops in the formation of anarray. Pulse jets operate by delivering a pulse of pressure (such as bya piezoelectric or thermoelectric element) to liquid adjacent an outletor orifice such that a drop will be dispensed therefrom.

A “set” of anything (such as a set of drops), may contain only one, oronly two, or three, or any number of multiple drops (although where“drops” are referenced in relation to a set implies the set in that caseincludes multiple drops). A “group” of drops has multiple drops. An“array”, unless a contrary intention appears, includes any one, two orthree dimensional arrangement of addressable regions bearing aparticular chemical moiety or moieties (for example, biopolymers such aspolynucleotide sequences) associated with that region. An array is“addressable” in that it has multiple regions of different moieties (forexample, different polynucleotide sequences) such that a region (a“feature” or “spot” of the array) at a particular predetermined location(an “address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). Array features are typically, but need not be, separated byintervening spaces. In the case of an array, the “target” will bereferenced as a moiety in a mobile phase (typically fluid), to bedetected by probes (“target probes”) which are bound to the substrate atthe various regions. However, either of the “target” or “target probes”may be the one which is to be evaluated by the other (thus, either onecould be an unknown mixture of polynucleotides to be evaluated bybinding with the other). An “array layout” refers collectively to one ormore physical, chemical or biological characteristics of the features,such as feature positioning, one or more feature dimensions, or someindication of an identity or function (for example, chemical orbiological) of a moiety at a given location. “Hybridizing” and“binding”, with respect to polynucleotides, are used interchangeably.During a “cycle” for forming a given feature, typically at least 50%(and more typically at least 70%, 80% or more preferably at least 90% or95%) of moieties bound to a substrate surface available to link with adeposited monomeric unit or previously obtained complete moiety forforming the desired feature, actually link to such deposited monomericunit or complete moiety.

When one item is indicated as being “remote” from another, this isreferenced that the two items are at least in different buildings, andmay be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data. An array “package” may be the array plus only asubstrate on which the array is deposited, although the package mayinclude other features (such as a housing with a chamber). A “chamber”references an enclosed volume (although a chamber may be accessiblethrough one or more ports). It will also be appreciated that throughoutthe present application, that words such as “top”, “upper”, and “lower”are used in a relative sense only. “Fluid” is used herein to reference aliquid. Reference to a singular item, includes the possibility thatthere are plural of the same items present. The steps of any method maybe performed in the recited order, or in any other order that islogically possible. For example, the depositing and detecting drops atrespective separate locations on the substrate from different dispenserswhich deposit a multi-dispenser drop group, may be performed before,during, or after array fabrication. All patents and other referencescited in this application, are incorporated into this application byreference except insofar as where any definitions in those referencesconflict with those of the present application (in which case thedefinitions of the present application are to prevail).

Referring first to FIGS. 1-3, typically methods and apparatus of thepresent invention generate or use a contiguous planar substrate 10carrying one or more arrays 12 disposed across a front surface 1 la ofsubstrate 10 and separated by inter-array areas 13. A back side 11 b ofsubstrate 10 does not carry any arrays 12. The arrays on substrate 10can be designed for testing against any type of sample, whether a trialsample, reference sample, a combination of them, or a known mixture ofpolynucleotides (in which latter case the arrays may be composed offeatures carrying unknown sequences to be evaluated). While ten arrays12 are shown in FIG. 5 and the different embodiments described below mayuse substrates with particular numbers of arrays, it will be understoodthat substrate 10 and the embodiments to be used with it, may use anynumber of desired arrays 12. Similarly, substrate 10 may be of anyshape, and any apparatus used with it adapted accordingly. Dependingupon intended use, any or all of arrays 12 may be the same or differentfrom one another and each will contain multiple spots or features 16 ofbiopolymers in the form of polynucleotides. A typical array may containfrom more than ten, more than one hundred, more than one thousand or tenthousand features, or even more than from one hundred thousand features.All of the features 16 may be different, or some or all could be thesame. In the case where arrays 12 are formed by the conventional in situor deposition of previously obtained moieties, as described above, bydepositing for each feature a droplet of reagent in each cycle such asby using a pulse jet such as an inkjet type head, interfeature areas 17will typically (but not essentially) be present which do not carry anypolynucleotide. It will be appreciated though, that the interfeatureareas 17 could be of various sizes and configurations. It will also beappreciated that there need not be any space separating arrays 12 fromone another. Each feature carries a predetermined polynucleotide (whichincludes the possibility of mixtures of polynucleotides). As per usual,A, C, G, T represent the usual nucleotides. It will be understood thatthere is usually a linker molecule (not shown) of any known typesbetween the front surface 11 a and the first nucleotide.

FIGS. 2 and 3 illustrate ideal features where the actual features formedare the same as the target (or “aim”) features, with each feature 16being uniform in shape, size and composition, and the features beingregularly spaced. Such an array when fabricated by drop depositionmethods, would require all reagent droplets for each feature to beuniform in shape and accurately deposited at the target featurelocation. In practice, such an ideal result is difficult to obtain dueto fixed and random errors during fabrication.

For the purposes of the discussions below, it will be assumed (unlessthe contrary is indicated) that the array being formed in any case is apolynucleotide array formed by the in situ method using pulse jetdispensers. However, the applicability of the method to arrays of otherpolymers or chemical moieties generally, whether formed by multiplecycle in situ methods or deposition of previously obtained moieties, orusing other procedures or types of dispensers, will be understood fromthese discussions.

FIGS. 4 and 5 illustrates in part a method of the present invention.Referring to FIG. 4, this is a top view of orifices 220 of dropdispensers in a dispensing head 210 (see below). That is, FIG. 4 islooking from above the head down onto a substrate 10 onto which dropsare to be deposited from orifices 220. Each column of three orifices220A, 220C, 220G, and 220T dispense a corresponding (that is, adenosine,cytidine, guanosine, thymidine, respectively) a drop of a solutioncontaining a nucleoside phosphoramidite which will only become attachedto a location at which it is deposited, upon activation by a suitableactivator. Suitable activators and related chemistry is described inthose references cited above relating to polynucleotide synthesis, aswell as in U.S. patent application Ser. No. 09/356,249 titled“Biopolymer Arrays And Their Fabrication” by Perbost, filed Jul. 16,1999. Another column of three orifices 220B will dispense a solutioncontaining the activator.

FIG. 5 schematically illustrates a deposited drop pattern on substrate10 over two cycles, for fabrication of the first two nucleotides of thepolynucleotides at the multiple locations of features 16 a, 16 b, 16 cto be formed as illustrated in FIG. 3. While only locations of features16 a, 16 b, 16 c will be discussed for simplicity, it will be understoodthat drops are deposited during each cycle at further feature locationsin an analogous manner. In a first cycle, at each of those locations(and at other feature 16 locations of an array 12) a reagent drop set isdeposited to attach a nucleoside monomer, and this procedure repeated atthose locations until the polynucleotides illustrated in FIG. 3 arefabricated. A multi-dispenser drop group is deposited at each of thelocations 16 a, 16 b, 16 c both with a single cycle and over multiplecycles. Specifically, in a first cycle orifices 220T, 220G, and 220Adeposit respective drops 240T, 240G, 240A of phosphoramidite monomersolution at locations 16 a, 16 b, 16 c, respectively on substrate 10(note in FIG. 5 deposited drops 240 are shown as only somewhatoverlapping for clarity, in practice the aim is to have all drops at afeature location overlap each other completely). This is followed, orpreceded, by dispensing of a drop 240B of activator solution from anorifice 220B, in the same cycle. As a result, a T, G, A monomer becomesattached to locations 16 a, 16 b, 16 c, respectively. All locations onsubstrate 10 may then be exposed simultaneously (by flowing across thesurface) to reagents for phosphite oxidation, deblocking, and optionallycapping, in a known manner. In a second cycle orifices 220G, 220C, 220Tdeposit a drop 240G, 240C, 240T of corresponding phosphoramiditecontaining monomer at locations 16 a, 16 b, 16 c, respectively. This isfollowed, or preceded, by dispensing of a drop of activator solutionfrom an orifice 220B, in the same cycle. Further cycles are performed asrequired at all feature locations for the array 12 until array 12 isfabricated.

Note that in the foregoing description of FIGS. 4 and 5, amulti-dispenser drop group during a given cycle consists of a drop ofone of the phosphoramidites deposited from orifice 220A, 220G, 220T,220C and a drop of activator from one of the orifices 220B. For example,drops 240A and 240B deposited in a same cycle at location 16 aconstitute a multi-dispenser drop during one cycle. It will beappreciated that in FIGS. 3 and 4 a multi-dispenser drop group overmultiple cycles may include drops from all those different dispenserswhich deposit drops at a single one of the locations 16 a, 16 b, 16 c.For example, a multi-dispenser drop group over two cycles at location 16a consists of drops 240A, 240B, 240G, 240B. However, dispensers whichdispense a multi-dispenser drop group need not necessarily dispensedifferent reagent compositions. For example, it may be desired todispense multiple same reagent composition drops from respectivedifferent dispensers in a same cycle to average any errors occurringfrom those dispensers. However, in FIGS. 4 and 5 at least one of thedrops of different multi-dispenser drop groups are deposited from a samedispenser. For example, over an entire array 12 many of themulti-dispenser drop groups will include at least one drop of a samecomposition which may be from a same one of the dispensers 220B(although other multi-dispenser drop groups will include a drop from adifferent one of the dispensers 220B). Furthermore, it will be seen fromthe foregoing that different multi-dispenser drop groups may have atleast one drop deposited by a same dispenser and another drop depositedby a different dispenser. For example, during the first cycle amulti-dispenser drop group for location 16 a consists of a drop from adispenser 220T and a particular one dispenser 220B, while amulti-dispenser drop group for location 16 b may consist of a drop fromdispenser 220G and the same particular one dispenser 240B. It will beappreciated that multiple drops of a same reagent from a same dispensercan be deposited during any one cycle (instead of just one drop asdescribed above).

In the present invention, drops deposited from different dispenserswhich deposit a multi-dispenser drop group may be deposited and detectedat separate locations on the substrate. For example, the dispenser 220Tand the dispenser 220B which deposit the multi-dispenser drop group 240Tand 240B during the first cycle at location 16 a, are each made todeposit at least one drop at separate different locations on substrate10. One simple way of doing this is to deposit a single drop from eachdispenser of FIG. 5 in a test pattern 250 as illustrated in FIG. 6. Thedrops deposited in test pattern 250 can be individually detected attheir respective test locations in pattern 250, whereas this may not bepossible in the feature locations of array 12. For example, drops 240T,240B of the multi-dispenser drop group deposited by dispensers 220T,220B during the first cycle at location 16 a will both contact eachother in the liquid state and will therefore mix. Thus, it is difficultto detect subsequently deposited drop 240B at location 16a in order tocheck for any error in the dispenser of orifice 220B which depositedthat drop. However, it will be easier to check for an error in thatdispenser 220B and the dispenser 220T by detecting the drops depositedby them in the separate locations in test pattern 250. Furthermore, thephosphoramidite monomer of any of drops 240A, 240C, 240G, 240T willbecome attached (either directly to the substrate 10 or to aphosphoramidite monomer attached to that location in the precedingcycle) at the location 16 to which it is deposited, since an activatorcontaining drop 240B is added to each location in each cycle. However,each phosphoramidite containing monomer drop 240A, 240C, 240G, 240T doesnot become attached at test pattern 250 since an activator containingdrop 240B is not deposited at the same locations as those monomercontaining drops. Reagents in test pattern 250 deposited during onecycle are therefore washed off during oxidation, deblocking, or cappingsteps preceding a next cycle, such that the same test pattern 250 areacan be readily used again for a next cycle.

Drops deposited at test locations need not be deposited at a separatepattern 250. For example, the test locations could be interspersedbetween feature locations 16 (that is, in FIG. 2 some of the features 16could instead be test locations). However, it may be convenient to havea separate test pattern 250. Any test pattern 250 described herein maybe variously located. In one arrangement illustrated in FIG. 7, each ofmultiple test patterns 250 are positioned between two arrays 12. Testpatterns 250 in FIG. 7 extend across the same area (represented by aline 260) through which substrate 10 is later cut or otherwise separatedto provide multiple portions of the substrate each with one or morefabricated arrays thereon. Substrate 10 can of course be cut along anadditional line perpendicular to lines 260 to provide just one array 12on a substrate portion, if desired.

FIG. 8 illustrates a portion of a particular test pattern. FIG. 8 islooking down from above in the same manner as FIGS. 4-6. In thisexample, head 210 has a total of six different regions 222 a through 222f, each of which has two columns of ten orifices 220 a through 220 tcommunicating with a common fluid reservoir (that is, a separate fluidreservoir communicates with a total of twenty orifices 220 a through 220t in respective pulse jets). Any four reservoirs (for example, thereservoirs for regions 222 a, 222 b, 222 d, 222 e) may containrespective different phosphoramidite monomer solutions with two others(for example, for regions 222 c, 222 f) containing activator solutions.Head 210 is moved to form a test pattern 250 which has six columns oftwenty locations, each column having a deposited drop 240 a through 240t deposited from respective orifices 220 a through 220 t of one headregion 222. In FIG. 6 only the two columns 242 a and 242 b deposited byhead regions 222 a and 222 b, respectively, are shown. It will beunderstood thought that there will be four more such columns 242deposited by the four additional regions 222 c through 222 f. Thespacing of the locations 240 in such a test pattern corresponds to thatof features 16 in the arrays 12 being formed. The particular testpattern 250 of FIG. 6 not only allows convenient detection of drops fromdispensers of a multi-dispenser drop group, but also tests the headpositioning software and hardware (due to re-positioning of thetwo-column format of a region 222 of head 210 into a single column 242format) and the ability of the dispensers to correctly space dispenseddrops at features 16 (since the spacing at test locations 240 is alsothe same, although it need not be so).

Referring now to FIG. 9, an apparatus of the present invention which canexecute a method of the present invention, will now be described. Theapparatus shown includes a substrate station 20 on which can be mounteda substrate 10. Pins or similar means (not shown) can be provided onsubstrate station 20 by which to approximately align substrate 10 to anominal position thereon (with alignment marks 18 on substrate 10 beingused for more refined alignment). Substrate station 20 can include avacuum chuck connected to a suitable vacuum source (not shown) to retaina substrate 14 without exerting too much pressure thereon, sincesubstrate 14 is often made of glass. A flood station 68 is providedwhich can expose the entire surface of substrate 10, when positionedbeneath station 68 as illustrated in broken lines in FIG. 4, to a fluidtypically used in the in situ process, and to which all features must beexposed during each cycle (for example, oxidizer, deprotection agent,and wash buffer). In the case of deposition of a previously obtainedpolynucleotide, flood station 68 need not be present.

A dispensing head 210 is retained by a head retainer 208. Thepositioning system includes a carriage 62 connected to a firsttransporter 60 controlled by processor 140 through line 66, and a secondtransporter 100 controlled by processor 140 through line 106.Transporter 60 and carriage 62 are used execute one axis positioning ofstation 20 (and hence mounted substrate 10) facing the dispensing head210, by moving it in the direction of arrow 63, while transporter 100 isused to provide adjustment of the position of head retainer 208 (andhence head 210) in a direction of axis 204. In this manner, head 210 canbe scanned line by line, by scanning along a line over substrate 10 inthe direction of axis 204 using transporter 100, while line by linemovement of substrate 10 in a direction of axis 63 is provided bytransporter 60. Transporter 60 can also move substrate holder 20 toposition substrate 10 beneath flood station 68 (as illustrated by thesubstrate 10 shown in broken lines in FIG. 4). Head 210 may alsooptionally be moved in a vertical direction 202, by another suitabletransporter (not shown). It will be appreciated that other scanningconfigurations could be used. It will also be appreciated that bothtransporters 60 and 100, or either one of them, with suitableconstruction, could be used to perform the foregoing scanning of head210 with respect to substrate 10. Thus, when the present applicationrecites “positioning” or “moving” one element (such as head 210) inrelation to another element (such as one of the stations 20 or substrate10) it will be understood that any required moving can be accomplishedby moving either element or a combination of both of them. The head 210,the positioning system, and processor 140 together act as the depositionsystem of the apparatus. An encoder 30 communicates with processor 140to provide data on the exact location of substrate station 20 (and hencesubstrate 10 if positioned correctly on substrate station 20), whileencoder 34 provides data on the exact location of holder 208 (and hencehead 210 if positioned correctly on holder 208). Any suitable encoder,such as an optical encoder, may be used which provides data on linearposition.

Processor 140 also has access through a communication module 144 to acommunication channel 180 to communicate with a remote station.Communication channel 180 may, for example, be a Wide Area Network(“WAN”), telephone network, satellite network, or any other suitablecommunication channel.

Head 210 may be of a type commonly used in an ink jet type of printerand may, for example, include five or more chambers (at least one foreach of four nucleoside phosphoramidite monomers plus at least one foran activator solution) each communicating with a corresponding set ofmultiple drop dispensing orifices and multiple ejectors which arepositioned in the chambers opposite respective orifices. Each ejector isin the form of an electrical resistor operating as a heating elementunder control of processor 140 (although piezoelectric elements could beused instead). Each orifice with its associated ejector and portion ofthe chamber, defines a corresponding pulse jet. It will be appreciatedthat head 210 could, for example, have more or less pulse jets asdesired (for example, at least ten or at least one hundred pulse jets).Application of a single electric pulse to an ejector will cause adroplet to be dispensed from a corresponding orifice. Certain elementsof the head 210 can be adapted from parts of a commercially availablethermal inkjet print head device available from Hewlett-Packard Co. aspart no. HP51645A. Alternatively, multiple heads could be used insteadof a single head 210, each being similar in construction to head 210 andbeing provided with respective transporters under control of processor140 for independent movement. In this alternate configuration, each headmay dispense a corresponding biomonomer (for example, one of fournucleoside phosphoramidites) or an activator solution.

As is well known in the ink jet print art, the amount of fluid that isexpelled in a single activation event of a pulse jet, can be controlledby changing one or more of a number of parameters, including the orificediameter, the orifice length (thickness of the orifice member at theorifice), the size of the deposition chamber, and the size of theheating element, among others. The amount of fluid that is expelledduring a single activation event is generally in the range about 0.1 to1000 pL, usually about 0.5 to 500 pL and more usually about 1.0 to 250pL. A typical velocity at which the fluid is expelled from the chamberis more than about 1 m/s, usually more than about 10 m/s, and may be asgreat as about 20 m/s or greater. As will be appreciated, if the orificeis in motion with respect to the receiving surface at the time anejector is activated, the actual site of deposition of the material willnot be the location that is at the moment of activation in aline-of-sight relation to the orifice, but will be a location that ispredictable for the given distances and velocities.

The apparatus can deposit droplets to provide features which may havewidths (that is, diameter, for a round spot) in the range from a minimumof about 10 μm to a maximum of about 1.0 cm. In embodiments where verysmall spot sizes or feature sizes are desired, material can be depositedaccording to the invention in small spots whose width is in the rangeabout 1.0 μm to 1.0 mm, usually about 5.0 μm to 500 μm, and more usuallyabout 10 μm to 200 μm.

The apparatus further includes a display 310, speaker 314, and operatorinput device 312. Operator input device 312 may, for example, be akeyboard, mouse, or the like. Processor 140 has access to a memory 141,and controls print head 210 (specifically, the activation of theejectors therein), operation of the positioning system, operation ofeach jet in print head 210, and operation of display 310 and speaker314. Memory 141 may be any suitable device in which processor 140 canstore and retrieve data, such as magnetic, optical, or solid statestorage devices (including magnetic or optical disks or tape or RAM, orany other suitable device, either fixed or portable). Processor 140 mayinclude a general purpose digital microprocessor suitably programmedfrom a computer readable medium carrying necessary program code, toexecute all of the steps required by the present invention, or anyhardware or software combination which will perform those or equivalentsteps. The programming can be provided remotely to processor 141 throughcommunication channel 180, or previously saved in a computer programproduct such as memory 141 or some other portable or fixed computerreadable storage medium using any of those devices mentioned below inconnection with memory 141. For example, a magnetic or optical disk 324a may carry the programming, and can be read by disk writer/reader 326.A cutter 152 is provided to cut substrate 10 into individual array units15 each carrying a corresponding array 12.

The operation of the fabrication station will now be described. It willbe assumed that a substrate 10 on which arrays 12 are to be fabricated,is in position on station 20 and that processor 140 is programmed withthe necessary layout information to fabricate target arrays 12. Usinginformation such as the foregoing target layout and the number andlocation of drop dispensers in head 210, processor 140 can thendetermine a reagent drop deposition pattern. Alternatively, such apattern could have been determined by another processor (such as aremote processor) and communicated to memory 141 through communicationchannel 180 or by forwarding a portable storage medium carrying suchpattern data for reading by reader/writer 326. Processor 140 controlsfabrication, in accordance with the deposition pattern, to generate theone or more arrays 12 on substrate 10 by depositing for each targetfeature during each cycle, a reagent drop set as previously described.Processor 140 also sends substrate 10 to flood station 68 for cycleintervening or final steps as required, all in accordance with theconventional in situ polynucleotide array fabrication process describedabove. The substrate 10 is then sent to a cutter 152 wherein portions ofsubstrate 10 carrying one ore more arrays 12 are separated from theremainder of substrate 10, to provide multiple array units 15 each withone or more arrays 12. One or more array units 15 may then be forwardedto one or more remote users. Processor 140 also causes deposition ofdrops from all multi-dispenser drop groups to be deposited at separatetest locations, such as at a test pattern 250 which may be separate fromarrays 12 as already described above.

Deposited drops at any test location can be detected by capturing animage of the drops such as described in U.S. patent applications:“Polynucleotide Array Fabrication” by Caren et al., Ser. No. 09/302,898filed Apr. 30, 1999; and “Biopolymer Array Inspection”, Ser. No.09/419,447, filed Oct. 15, 1999 by Fisher. Alternatively, drops can bedetected by using the method described in U.S. patent application “ArrayFabrication With Drop Detection”, Ser. No. 09/558,532, filed Apr. 26,2000 by Schantz et al. When an error is sensed in a dispenser,fabrication can be halted and an attempt made to correct the error (forexample, by cleaning or replacing the dispenser in error). As well, oneor more arrays 12 which will likely contain an error in one or morefeatures 16 as a result of the detected dispenser error, can bediscarded or further drops can be deposited from one or more dispensersfrom the same or a replacement head 210 in an attempt to correct thearray errors. Alternatively, or additionally, results from the detection(whether raw or processed in some way) or any such identified errors inthe array, can be saved in a local memory at a fabrication station inassociation with a file and an assigned identifier, and the identifierassociated with the array and forwarded to a remote end user station, inany of those manners as described in U.S. patent application Ser. No.09/302,898 mentioned above. For example, the identifier may be appliedto the array substrate 10 or a housing carrying it, and array andassociated identifier forwarded to the remote user station. In avariation, the results or identified errors can be saved on a portablestorage medium which is shipped to the end user, typically inassociation with the array (for example, in the same package), asfurther described in the foregoing application.

The results from the detection may be used alternatively or additionallyin a number of ways. In one way, the results from the detecting may beevaluated based at least in part on a cycle during which the resultswere obtained. Depending upon the particular chemistry used in thefabrication process, errors in the location of drop depositions may begiven greater weight as a function of the cycle during which they occur.For example, for the polynucleotide in situ fabrication usingphosphoramidite chemistry, errors in drop location during later cyclesmay be assigned less weight than an error of the same magnitude in anearlier cycle (such as in the first cycle). Also, a parameter (such aslocation or size) of the dispensing of the drops to fabricate the array,may be adjusted before or during array fabrication (or after arrayfabrication with the results being used for fabricating a subsequentarray) based at least in part on the results of the detecting. Forexample, dispensers or their positioning may be calibrated based on suchresults. In another application, for the drops detected replicates of asame drop from a same dispenser may be deposited at multiple differentlocations on the substrate. In this case, a characteristic of thesubstrate may be evaluated based on the results of detecting thereplicates. For example, if the dispensers are known to function todeposit drops reproducibly, then if detected deposited drops have adifferent size on the substrate, this would indicate the substratesurface does not have homogeneous properties. Such a substrate qualityassessment may be performed before, during, or after array fabrication.In a further application, dispenser performance may be evaluated basedon relative characteristics of drops of different composition depositedfrom different dispensers. For example, the activator tetrazole has aviscosity (typically approximately 4 cp) which is much less than aphosphoramidite viscosity (about 7 cp) and thus a deposited tetrazoledrop tends to have a larger diameter on the substrate than a depositeddrop of the same volume of phosphoramidite. Where the detecting includesa measurement of the size of the area covered by a deposited drop, theresults from detecting could be used to evaluate for errors (such asload or prime errors). Note that other components could be included inthe reagents to further distinguish one from the other.

The above array fabrication sequence can be repeated at the fabricationstation as desired for multiple substrates 10 in turn. As mentionedabove, the fabrication station may act as a central fabrication stationfor each of multiple remote user stations, in the same manner asdescribed above. When a user receives an array unit 15, it willtypically be exposed to a sample and the array read following exposure.Array reading is usually accomplished by a suitable scanner which canread the location and intensity of fluorescence at each feature of anarray following exposure to a fluorescently labeled sample. For example,such a scanner may be similar to the GENEARRAY scanner available fromHewlett-Packard, Palo Alto, Calif. At the user station the any arraylayout information can be retrieved for an array 12 over a communicationchannel (or alternatively from a local memory) from the now remotememory at the fabrication station, in response to communicating theidentifier for that array. This retrieved information can be used in thereading or processing of read results from the array. The foregoingmethods are described in more detail in the previously mentioned Ser.No. 09/302,898. Results from reading can be processed such as byrejecting a reading for a feature which is below a predeterminedthreshold and/or forming conclusions based on the pattern read from thearray (such as whether or not a particular target sequence may have beenpresent in the sample). The results of the interrogation or processingcan be communicated for receipt at a remote location if desired, forfurther use.

In a variation of the above, it is possible that each unit 15 may becontained with a suitable housing. Such a housing may include a closedchamber accessible through one or more ports normally closed by septa,which carries the substrate 10.

Modifications in the particular embodiments described above are, ofcourse, possible. For example, where a pattern of arrays is desired, anyof a variety of geometries may be constructed other than the organizedrows and columns of arrays 12 of FIG. 1. For example, arrays 12 can bearranged in a series of curvilinear rows across the substrate surface(for example, a series of concentric circles or semi-circles of spots),and the like. Similarly, as mentioned, the pattern of features 16 may bevaried from the organized rows and columns of spots in FIG. 2 toinclude, for example, a series of curvilinear rows across the substratesurface(for example, a series of concentric circles or semi-circles ofspots), and the like.

The present methods and apparatus may be used to deposit biopolymers orother moieties on surfaces of any of a variety of different substrates,including both flexible and rigid substrates. Preferred materialsprovide physical support for the deposited material and endure theconditions of the deposition process and of any subsequent treatment orhandling or processing that may be encountered in the use of theparticular array. The array substrate may take any of a variety ofconfigurations ranging from simple to complex. Thus, the substrate couldhave generally planar form, as for example a slide or plateconfiguration, such as a rectangular or square or disc. In manyembodiments, the substrate will be shaped generally as a rectangularsolid, having a length in the range about 4 mm to 200 mm, usually about4 mm to 150 mm, more usually about 4 mm to 125 mm; a width in the rangeabout 4 mm to 200 mm, usually about 4 mm to 120 mm and more usuallyabout 4 mm to 80 mm; and a thickness in the range about 0.01 mm to 5.0mm, usually from about 0.1 mm to 2 mm and more usually from about 0.2 to1 mm. However, larger substrates can be used, particularly when such arecut after fabrication into smaller size substrates carrying a smallertotal number of arrays 12. Substrates of other configurations andequivalent areas can be chosen. The configuration of the array may beselected according to manufacturing, handling, and use considerations.

The substrates may be fabricated from any of a variety of materials. Incertain embodiments, such as for example where production of bindingpair arrays for use in research and related applications is desired, thematerials from which the substrate may be fabricated should ideallyexhibit a low level of non-specific binding during hybridization events.In many situations, it may also be preferable to employ a material thatis transparent to visible and/or UV light. For flexible substrates,materials of interest include: nylon, both modified and unmodified,nitrocellulose, polypropylene, and the like, where a nylon membrane, aswell as derivatives thereof, may be particularly useful in thisembodiment. For rigid substrates, specific materials of interestinclude: glass; fused silica, silicon, plastics (for example,polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like); metals (for example, gold, platinum, andthe like).

The substrate surface onto which the polynucleotide compositions orother moieties is deposited may be porous or non-porous, smooth orsubstantially planar, or have irregularities, such as depressions orelevations. The surface may be modified with one or more differentlayers of compounds that serve to modify the properties of the surfacein a desirable manner. Such modification layers, when present, willgenerally range in thickness from a monomolecular thickness to about 1mm, usually from a monomolecular thickness to about 0.1 mm and moreusually from a monomolecular thickness to about 0.001 mm. Modificationlayers of interest include: inorganic and organic layers such as metals,metal oxides, polymers, small organic molecules and the like. Polymericlayers of interest include layers of: peptides, proteins, polynucleicacids or mimetics thereof (for example, peptide nucleic acids and thelike); polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated).

Various further modifications to the particular embodiments describedabove are, of course, possible. Accordingly, the present invention isnot limited to the particular embodiments described in detail above.

1. A method of forming an addressable array of chemical moieties on asubstrate, comprising: (a) for each of multiple locations on thesubstrate, depositing a reagent drop set during a cycle so as to attacha corresponding moiety for that location; and (b) repeating step (a) ifrequired, until the addressable array is formed; wherein, for each ofmultiple locations, a multi-dispenser drop group is deposited over oneor more cycles for a corresponding location which group includes dropswhich are deposited from different dispensers; the method additionallycomprising: (c) depositing and detecting drops at respective separatelocations on the substrate from different dispensers which deposit amulti-dispenser drop group.
 2. A method according to claim 1 whereindrops of the multi-dispenser drop group in step (c) are notindependently detected at the corresponding location in step (b).
 3. Amethod according to claim 1 wherein a multi-dispenser drop groupcomprises a drop including an attachment moiety which becomes attachedat the location at which the group is deposited in step (a) or (b) butwhich does not become attached at a location in step (c).
 4. A methodaccording to claim 1 wherein a multi-dispenser drop group comprises adrop including an attachment moiety which will become attached at thelocation at which the group is deposited upon activation by anactivator, and at least one other drop comprises the activator moiety,such that the attachment moiety and activator are deposited at separatelocations in step (c).
 5. A method according to claim 1 wherein dropsare deposited and detected at respective separate locations on thesubstrate from all those dispensers which deposit a multi-dispenser dropgroup.
 6. A method according to claim 2 wherein in step (c) the dropsare detected on the substrate.
 7. A method according to claim 1additionally comprising capturing an image of drops deposited duringstep (c).
 8. A method according to claim 6 additionally comprisingevaluating results from the detecting for an indication of a dispensererror and, when an error is detected, discarding the array or depositingfurther drops to correct the error.
 9. A method according to claim 6additionally comprising saving results from the detecting in a memory.10. A method according to claim 6 additionally comprising evaluatingresults from the detecting based at least in part on a cycle duringwhich the results were obtained.
 11. A method according to claim 10wherein results from detecting during multiple cycles are obtained andthe evaluation is based at least in part on the cycles during which theresults were obtained.
 12. A method according to claim 2 additionallycomprising adjusting a parameter of the dispensing in step (a) based atleast in part on the results from step (c).
 13. A method according toclaim 1 wherein in step (c) replicates of a same drop from a samedispenser are deposited at multiple different locations on thesubstrate, the method additionally comprising evaluating acharacteristic of the substrate based on the results of detecting thereplicates.
 14. A method according to claim 1 additionally comprisingevaluating dispenser performance based on relative characteristics ofdrops of different composition deposited from different dispensers. 15.A method according to claim 1 wherein during step (a) or (b) drops ofmulti-dispenser drop groups are deposited at respective substratelocations such that one drop of the group contacts a previouslydeposited drop of the same group at the same location.
 16. A methodaccording to claim 2 wherein different multi-dispenser drop groups haveat least one drop deposited by a same dispenser and another dropdeposited by a different dispenser.
 17. A method according to claim 2wherein the at least some of the drops of a multi-dispenser drop set areof a different composition.
 18. A method according to claim 2 wherein atleast one of the drops of different multi-dispenser drop groups aredeposited from a same dispenser.
 19. A method according to claim 1wherein different multi-dispenser drop groups are deposited atrespective substrate locations in step (a) or (b), and wherein the dropsdeposited and detected in step (c) are deposited in a test pattern areaseparate from the array.
 20. A method of forming an addressable array ofchemical moieties on a substrate, comprising: (a) for each of multiplelocations on the substrate, depositing a reagent drop set during a cycleso as to attach a corresponding moiety for that location; and (b)repeating step (a) if required; wherein, for each of multiple locations,a multi-dispenser drop group is deposited over one or more cycles for acorresponding location which group includes drops which are depositedfrom different dispensers; the method additionally comprising: (c)depositing and detecting drops from different dispensers which deposit amulti-dispenser drop group, onto the substrate at respective separatelocations in a test pattern area separate from the array.
 21. A methodaccording to claim 20 wherein a multi-dispenser drop group comprises adrop including an attachment moiety which becomes attached at thelocation at which the group is deposited in step (a) or (b) but whichdoes not become attached at a location in step (c).
 22. A methodaccording to claim 20 wherein a multi-dispenser drop group comprises adrop containing an attachment moiety which will become attached at thatlocation upon activation by an activator, and at least one other dropcontaining the activator moiety, such that the attachment moiety andactivator are deposited at separate locations in step (c).
 23. A methodaccording to claim 22 wherein in step (c) no activator containing dropis deposited at a same location as an attachment moiety containing drop.24. A method according to claim 22 wherein different multi-dispenserdrop groups are deposited at respective substrate locations in step (a)or (b), and wherein drops from dispensers which deposit differentmulti-dispenser drop groups are deposited and detected in step (c) in atest pattern area separate from the array.
 25. A method of forming anaddressable array of polymers on a substrate, comprising: (a) for eachof multiple locations on the substrate, depositing a reagent drop setduring a cycle so as to attach a monomeric unit of the correspondingpolymer for that location; and (b) repeating step (a), until theaddressable array is formed; wherein, for each of multiple locations, amulti-dispenser drop group is deposited over one or more cycles for acorresponding location which group includes drops which are depositedfrom different dispensers; the method additionally comprising: (c)depositing and detecting drops at respective separate locations on thesubstrate from different dispensers which deposit a multi-dispenser dropgroup.
 26. A method according to claim 25 wherein a multi-dispenser dropgroup comprises a drop including an attachment moiety which becomesattached at the location at which the group is deposited in step (a) or(b) but which does not become attached at a location in step (c).
 27. Amethod according to claim 25 wherein the polymers are biopolymers.
 28. Amethod according to claim 27 wherein a multi-dispenser drop groupdeposited during a cycle comprises a drop including the monomeric unitwhich will become attached at that location upon activation by anactivator, and at least one other drop comprises the activator moiety,such that the monomeric unit and activator are deposited at separatelocations in step (c).
 29. A method according to claim 25 wherein step(c) is performed between two cycles.
 30. A method according to claim 25wherein step (c) is performed between two cycles, and performed againbetween another two cycles.
 31. A method according to claim 25 whereindrops are deposited and detected at respective separate locations on thesubstrate from all those dispensers which deposit a multi-dispenser dropgroup.
 32. A method according to claim 25 wherein in step (c) the dropsare detected on the substrate.
 33. A method according to claim 25additionally comprising capturing an image of drops deposited duringstep (c).
 34. A method according to claim 28 wherein step (c) isperformed between two cycles, the method additionally comprising when anerror in a monomeric unit or activator drop dispenser is detected thendepositing further drops containing the monomeric unit or activator soas to correct the error.
 35. A method according to claim 26 whereinduring step (a) or (b) drops of multi-dispenser drop groups aredeposited at respective substrate locations such that one drop of thegroup contacts a previously deposited drop of the same group at the samelocation.
 36. A method according to claim 28 wherein the activatorcontaining drop for multiple locations is deposited from a samedispenser.
 37. A method according to claim 25 wherein differentmulti-dispenser drop groups are deposited at respective substratelocations in step (a) or (b), and wherein the drops deposited anddetected in step (c) are deposited in a test pattern area separate fromthe array.
 38. A method of forming multiple addressable arrays ofchemical moieties on a substrate, comprising for each array: (a) foreach of multiple locations on the substrate, depositing a reagent dropset during a cycle so as to attach a corresponding moiety for thatlocation; and (b) repeating step (a) if required, until the addressablearray is formed; wherein multiple dispensers are used to dispense dropsto form the array, the method additionally comprising: (c) depositingand detecting drops from the different dispensers at respective separatelocations on the substrate, wherein the drops are deposited at aseparate test pattern area between arrays with the number of locationsof the test pattern area during any one cycle being less than one tenththe number of locations in the smallest of the arrays which the testpattern area is between.
 39. A method according to claim 38 wherein thenumber of locations of the test pattern area during any one cycle is notgreater than ten times the number of the dispensers used to form anarray during any one cycle.
 40. An apparatus for forming an addressablearray on a substrate, comprising: (a) a deposition system havingmultiple dispensers each of which can dispense a reagent drop; (b) atransport system to move at least one of the deposition system or thesubstrate; (c) a drop detector; (d) a processor which controls thedeposition system and the transport system such that: (i) for each ofmultiple locations on the substrate, a reagent drop set is depositedduring a cycle so as to attach a corresponding moiety for that location;and (ii) step (i) will be repeated if required, until the addressablearray is formed; wherein, for each of multiple locations, amulti-dispenser drop group is deposited over one or more cycles for acorresponding location which group includes drops which are depositedfrom different dispensers and which drops are not independently detectedat the corresponding location; and such that: (iii) drops from differentdispensers which deposit a multi-dispenser drop group will be depositedand detected by the detector at respective separate locations on thesubstrate.
 41. An apparatus according to claim 40 wherein differentmulti-dispenser drop groups are deposited at respective substratelocations in step (i) or (ii), and wherein the drops deposited anddetected in step (iii) are deposited in a test pattern area separatefrom the array.
 42. An apparatus according to claim 40 wherein drops aredeposited and detected at respective separate locations on the substratefrom all those dispensers which deposit a multi-dispenser drop group.43. A method according to claim 40 wherein the detector detects drops onthe substrate.
 44. A method according to claim 40 wherein the detectorcaptures an image of deposited drops.
 45. A method according to claim 40wherein at least one of the drops of different multi-dispenser dropgroups are deposited from a same dispenser.
 46. An apparatus for formingan addressable array on a substrate, comprising: (a) a deposition systemhaving multiple dispensers each of which can dispense a reagent drop;(b) a transport system to move at least one of the deposition system orthe substrate; (c) a drop detector; (d) a processor which controls thedeposition system and the transport system such that: (i) for each ofmultiple locations on the substrate, a reagent drop set is depositedduring a cycle so as to attach a corresponding moiety for that location;and (ii) step (i) will be repeated if required, until the addressablearray is formed; wherein, for each of multiple locations, amulti-dispenser drop group is deposited over one or more cycles for acorresponding location which group includes drops which are depositedfrom different dispensers and which drops are not independently detectedat the corresponding location; and such that: (iii) drops from differentdispensers which deposit a multi-dispenser drop group will be depositedand detected by the detector at respective separate locations on thesubstrate in a test pattern area separate from the array.
 47. Anapparatus according to claim 46 wherein different multi-dispenser dropgroups are deposited at respective substrate locations in step (i) or(ii), and wherein drops from dispensers which deposit differentmulti-dispenser drop groups are deposited and detected in step (iii) ina test pattern area separate from the array.
 48. An apparatus forforming an addressable array on a substrate, comprising: (a) adeposition system having multiple dispensers each of which can dispensea reagent drop; (b) a transport system to move at least one of thedeposition system or the substrate; (c) a drop detector; (d) a processorwhich controls the deposition system and the transport system such that:(i) for each of multiple locations on the substrate, a reagent drop setis deposited during a cycle so as to attach a corresponding moiety forthat location; and (ii) repeating step (i) until the addressable arrayis formed; wherein, for each of multiple locations, a multi-dispenserdrop group is deposited over one or more cycles for a correspondinglocation which group includes drops which are deposited from differentdispensers; and such that: (iii) drops from different dispensers whichdeposit a multi-dispenser drop group will be deposited and detected bythe detector at respective separate locations on the substrate.
 49. Anapparatus according to claim 48 wherein the multi-dispenser drop groupis deposited during a cycle.
 50. A apparatus according to claim 49wherein step (i) is performed between two cycles.
 51. A apparatusaccording to claim 49 wherein step (i) is performed between two cycles,and performed again between another two cycles.
 52. A apparatusaccording to claim 49 wherein drops are deposited and detected atrespective separate locations on the substrate from all those dispenserswhich deposit a multi-dispenser drop group.
 53. A apparatus according toclaim 49 wherein during step (i) or (ii) drops of multi-dispenser dropgroups are deposited at respective substrate locations such that onedrop of the group contacts a previously deposited drop of the same groupat the same location.
 54. A computer program product, comprising: acomputer readable storage medium having a computer program storedthereon which, when loaded into a computer communicating with anapparatus for forming an addressable array on a substrate, performs thesteps of: (a) for each of multiple locations on the substrate,depositing a reagent drop set during a cycle so as to attach acorresponding moiety for that location; and (b) repeating step (a) ifrequired, until the addressable array is formed; wherein, for each ofmultiple locations, a multi-dispenser drop group is deposited over oneor more cycles for a corresponding location which group includes dropswhich are deposited from different dispensers and which drops are notindependently detected at the corresponding location; the stepsadditionally including: (c) depositing and detecting drops at respectiveseparate locations on the substrate from different dispensers whichdeposit a multi-dispenser drop group.
 55. A computer program productaccording to claim 54, wherein different multi-dispenser drop groups aredeposited at respective substrate locations in step (a) or (b), andwherein the drops deposited and detected in step (c) are deposited in atest pattern area separate from the array.
 56. A computer programproduct according to claim 55 wherein step (c) is performed between twocycles.
 57. A computer program product according to claim 55 wherein amulti-dispenser drop group comprises a drop including an attachmentmoiety which will become attached at that location upon activation by anactivator, and at least one other drop comprises the activator moiety,such that the attachment moiety and activator are deposited at separatelocations in step (c).
 58. A computer program product according to claim57 wherein in step (c) no activator containing drop is deposited at asame location as an attachment moiety containing drop.